For the purification (i.e. separation from impurities) and analysis of proteins and peptides (polypeptides), chromatography is a well-known and widely used method. A number of different chromatographic principles are applied, among these reversed phase high performance liquid chromatography (RP-HPLC). The RP-HPLC separation principle is based on hydrophobic association between the polypeptide solute and hydrophobic ligands on the chromatographic resin surface. RP-HPLC purification usually consists of one or more of the following sections: equilibration, loading, wash, elution, and regeneration.
Some impurities are poorly separated by traditional RP-HPLC carried out with a liquid phase having a pH lower or higher than the pl of the target molecule to be purified. Elution at pl is normally not the preferred mode of operation due to risk of uncontrolled precipitation of the product (i.e. peptide of interest). A pH gradient for separation of peptides was disclosed in ScienceDirect, Talanta 75 (2008), p. 76-82. Here a recurring pH gradient by repeating the same pH gradient from 2.5 to 10.5 was applied.
Various preparative reverse phase chromatography methods have been described. US2009036652 is related to purification of proteins using preparative reverse phase chromatography, WO2007071767 is related to purification of vitamin K-dependent polypeptides using preparative reverse phase chromatography and US20060211616 is related to purification of glucagon like peptides.
An optimized RP-HPLC purification method is however needed wherein good purification is obtained without the need for using an extensive process comprising a recurring pH gradient.